Biologically pure culture NRRL-B18737 useful in a method for biodegrading and biotransforming bisphenol alkanes, and bisphenol alkyl alcohols made therefrom

ABSTRACT

A gram-negative, aerobic, motile, rod-shaped bacterium which is closely related to Chryseomonas luteola and Flavimonas oryzihabitans, has been found to catalyze the biodegradation and biotransformation of bisphenol alkanes, such as 2,2-bis(4-hydroxyphenyl)-propane or bisphenol A. A variety of bisphenol alkyl alcohols can be made by this procedure.

This application is a continuation of application Ser. No. 07/625,381, filed Dec. 11, 1990, now U.S. Pat. No. 5,070,017.

BACKGROUND OF THE INVENTION

The present invention relates to a method for biotransforming bisphenol alkanes, such as bisphenol A, to convert these materials to hydroxylated reaction products, such as the corresponding bisphenol alkyl alcohols. The present invention also includes the biodegradation of bisphenol alkanes to carbonyl reaction products, such as aromatic aldehydes, aromatic acids, aromatic ketones and carbon dioxide.

Bisphenol alkanes, such as bisphenol A, are used in the production of plastics at numerous chemical manufacturing sites throughout the world. For example, annual production of bisphenol A exceeded 930M pounds in recent years. As a result, significant quantities of waste containing bisphenol A are generated at these various manufacturing facilities which is discharged into the terrestrial, aquatic and marine environments. According to standard evaluation procedures published by the US Environmental Protection Agency (EPA), bisphenol A was determined to be slightly to moderately toxic to fish and invertebrates. As a result, continuous efforts are being made to determine satisfactory procedures for minimizing the risks of bisphenol alkanes introduced into the environment.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that a certain gram-negative, aerobic, motile, rod-shaped bacterium, which is most closely related to Chryseomonas luteola and Flavimonas oryzihabitans, hereinafter referred to as strain MVl, has been found to catalyze the biodegradation and biotransformation of bisphenol alkanes. Suprisingly, strain MVl also has been found to selectively hydroxylate the aliphatic moiety of the bisphenol alkane resulting in the production of a variety of a trifunctional and tetrafunctional bisphenols which are useful as monomers for making plastics.

STATEMENT OF THE INVENTION

There is provided by the present invention, a method for biodegrading or biotransforming bisphenol alkanes, which comprises treating bisphenol alkanes under aerobic conditions at a pH in the range from 6.0 to 7.0 with an effective amount of a biologically pure culture of the gramnegative, motile, rod-shaped bacterium, NRRL-B-l8737 (or strain MVl).

In another aspect of the present invention, there is provided a method for biotransforming particular bisphenol alkanes to bisphenol alkyl alcohols by treating the bisphenol alkanes under aerobic conditions and at a pH in the range from 6.0 to 7.0 with an effective amount of a biologically pure culture of the gram-negative, motile, rod-shaped bacterium, NRRL-B-l8737, in an aqueous mineral salts medium at 25°-30°C.

A culture of the bacterial strain MVl used in the practice of the present invention, is on deposit with the U.S. Department of Agriculture as NRRL-B-l8737. A progeny of the subject microorganism will be provided by the assignee of this application under the conditions imposed by 37 CFR 1.114 and 35 USC 122 in the event that the Commissioner of Patents and Trademarks determines that an individual is entitled to the same.

Upon issuance of the subject application as a patent, a culture of this strain can be obtained from the permanent collection of the Agriculture, Peoria, IL., U.S.A.

The taxonomic identification of strain MVl is found to share features common to Chryseomonas luteola and Flavimonas oryzihabitans. Colonies of strain MVl were found to be round, smooth, entire and dull with a yellow pigment. Cells were found to be gram-negative rods with 1-2 polar flagella.

    ______________________________________                                         Key Biochemical Characteristics of Strain MV1                                  ______________________________________                                         Glucose              Acid (A)                                                  Fructose             A                                                         Mannose              A                                                         Xylose               A                                                         Mannitol             -                                                         MacConkey agar       growth                                                    Yellow pigment       +                                                         Gluconate oxidation  -                                                         Indole               -                                                         H.sub.2 S production -                                                         Oxidase              -                                                         Lysine               -                                                         Ornithine            -                                                         Lecithinase          -                                                         Hemolysis            Beta                                                      Gram positive        -                                                         Gram negative        +                                                         Gram variable        -                                                         Motile at 37 C.      -                                                         Motile at Room Temperature                                                                          +                                                         Flagella peritrichous                                                                               -                                                         Flagella lophotrichous                                                                              ?                                                         Flagella monotrichous                                                                               +                                                         Flagella lateral     -                                                         4° C. growth  -                                                         25° C. growth +                                                         30° C. growth +                                                         37° C. growth +                                                         41° C. growth -                                                         Pigment diffusible   -                                                         Pigment non-diff.    +                                                         Pyocyanine produced  -                                                         Fluorescein produced -                                                         Melanin pigment produced                                                                            -                                                         pH 6.0 growth        +                                                         3% NaCl growth       +                                                         6.5% NaCl growth     -                                                         Skim milk agar growth                                                                               +                                                         Aesculin hydrolysis  +                                                         Casein hydrolysis    -                                                         Starch hydrolysis    +                                                         Gelatinase           -                                                         Tween 80 hydrolysis  +                                                         Simmons citrate growth                                                                              +                                                         Urease               W                                                         Nitrate to nitrite   -                                                         Nitrite reduction    -                                                         Hydrogen sulfide (TSI) PbAc                                                                         +                                                         Lysine decarboxylase -                                                         Arginine (Mollers)   -                                                         Ornithine decarboxylase                                                                             -                                                         Phenylalanine deamination                                                                           -                                                         Phosphatase          +                                                         Catalase             +                                                         Growth on malonate as SCS                                                                           -                                                         Tyrosine degradation W                                                         dl - hydroxybutyrate growth                                                                         +                                                         PHB accumulation     +                                                         Deoxyribonuclease    -                                                         Growth on 0.05% cetrimide                                                                           -                                                         Growth on acetate as SCS                                                                            +                                                         Testosterone deg.    -                                                         3-Ketolactose        -                                                         ______________________________________                                    

Some of the bisphenol alkanes which can be hydroxylated in accordance with the practice of the invention are, for example, ##STR1##

The biochemical pathway for the biodegradation of bisphenol A by strain MVl is as follows: ##STR2##

In order that those skilled in the art will be better able to practice the present invention, the following examples are given by way of illustration and not by way of limitation. All parts are by weight unless otherwise indicated.

EXAMPLE 1

Ten ml of a sludge containing bisphenol A taken from an industrial waste treatment plant was added to 50 ml of a PAS culture medium containing 100 mg of bisphenol A and 0.005% yeast extract. The PAS culture medium consisted of the following:

    ______________________________________                                         SALT           g/L      Final conc. g/L medium                                 ______________________________________                                         PAS Salts (100×)                                                         MgSO.sub.4     19.5   g     0.195   g/L                                        MnSO.sub.4.H.sub.2 O                                                                          5.0          0.05                                               FeSO.sub.4.7H.sub.2 O                                                                         1.0          0.01                                               CaCl.sub.2.2H.sub.2 O                                                                         0.3          0.03                                               conc. HCL--1 ml/L                                                              Filter Sterilized                                                              PA Concentrate (20×)                                                     K.sub.2 HPO.sub.4                                                                             87.0   g     4.35                                               KH.sub.2 PO.sub.4                                                                             33.7         1.69                                               NH.sub.4 Cl    52.5         2.13                                               ______________________________________                                          *PA added before or after autoclaving, depending on substrate.                 ADD 100× PAS salts after autoclaving                                     *For agar plates, use 15 g purified agar/L of medium.                          *Final conc. is 37 mM phosphate, pH = 6.8                                

The resulting mixture was incubated aerobically under sealed conditions in a rotary shaker-incubator set at 30° C. and 200 RPM. After one week, the bisphenol A particles disappeared from the culture medium. A subculture of the week-old culture was obtained by transferring 2 ml of the grown enrichment culture to 50 ml of PAS medium containing bisphenol A (100 mg) as the carbon source. After another week, a second subculture was obtained by transferring 2 ml of the 1st subculture into another 50 ml PAS medium with bisphenol A. The second subculture grew to an optical density of 2.5 (absorbance at 600 nm, spectronic 20 spectrophotometer) after 48 hours incubation. The subculture was used to study the rate of bisphenol A degradation as a mixed culture via subsequent subcultures.

A sample of the above enriched subculture was then grown on PAS plates utilizing a PAS medium solidified with 1.5% agar which contained fine crystals of bisphenol A dispersed throughout a culture dish. The enriched subculture which was grown on bisphenol A was streaked out on the PAS plates and incubated for 2 weeks. After one week, the growth which resulted did not produce any isolated colonies. However, a clearing of the agar due to the dissolution and degradation of bisphenol A was observed around and under the area where cell growth occurred. Upon restreaking several times on PAS plates containing bisphenol A, isolated colonies were obtained. The isolated colonies were checked for uniformity on a nutritionally-rich medium containing glucose as a carbon and energy source. The colonies which resulted were demonstrated to be uniform and of a single species of bacterium (strain MVl). Poor growth was observed when the bacterial isolates were returned to PAS medium with bisphenol A as the sole carbon source. The strain MVl had a tendency to lose its ability to grow on bisphenol A at saturating levels when it was cultured on alternate substrates in the absence of bisphenol A for extended periods of time.

A carbon mass balance for strain MVl grown on bisphenol A was done utilizing a total organic carbon analyzer (Shimadzu, Japan). Strain MVl was grown in triplicate sealed 120 ml serum bottles containing 20 ml of PAS medium and 0.132 millimoles bisphenol A (equal to 2 millimoles carbon). After inoculation, the serum bottles were pressurized with air to approximately 1.5 atmospheres and incubated at 30° C. on a rotary shaker incubator. Complete degradation of the bisphenol A resulted after 48 hours. Almost 60% of the carbon in bisphenol A was mineralized to carbon dioxide (CO₂) and 20% was associated with the bacterial cells. The remaining 20% of the carbon was recovered in the culture medium as soluble organic carbon after acidification to remove the CO₂. The following table shows the results obtained:

    ______________________________________                                         CARBON MASS BALANCE FOR THE BIO-                                               DEGRADATION OF BISPHENOL A BY BACTERIAL                                        STRAIN MVI                                                                     SUBSTRATE         PRODUCTS                                                     (mMoles C)        (mMoles C)                                                   Cul-                                    Total C                                ture  Carbon in  Carbon   Carbon Carbon Re-                                    Bottle                                                                               Bisphenol A                                                                               Dioxide  Biomass                                                                               Aqueous                                                                               covered                                ______________________________________                                         1     2.00       1.23     0.37   0.40   2.00                                   2     2.00       1.21     0.40   0.42   2.03                                   3     2.00       1.04     0.40   0.43   1.87                                   Average of 3 Bottles                                                                        1.16     0.39     0.42   1.97                                     Percent of Total                                                                             59%     19.8%    21.4%  98.3%                                    Carbon                                                                         ______________________________________                                    

Above results show that 80% of the bisphenol A is degraded to CO₂ or converted to biomass. The remaining 20% of the bisphenol is biotransformed to soluble bisphenol A hydroxylation products.

EXAMPLE 2

In accordance with the procedure of example 1, there was added 5 volume % of a culture of strain MVl grown on bisphenol A to one liter of PAS medium at 30° C. containing 5 grams of bisphenol A. The mixture was incubated for 4-5 days at 30° C. under aerobic conditions with limiting oxygen at a pH of 6.0-7.0. HPLC showed the formation of 2,2-bis(4-hydroxyphenyl)-1-propanol up to a concentration of 1.5 grams per liter.

EXAMPLE 3

Several bisphenol alkanes were individually incubated over a period of a few hours to several days at ambient temperatures with 1-2 grams wet weight of viable strain MVl cells, which were grown on bisphenol A in accordance with the procedure of example 1. The following hydroxylation products were obtained. ##STR3##

The above results show that the method of the present invention provides a procedure for making a variety of bisphenol alcohols which are useful compounds for making thermoplastics, such as polycarbonates and polyesters.

Although the above examples are directed to only few of the very many variables which can be employed in the practice of the present invention, it should be understood that the present invention is directed to a much broader variety of bisphenol alkanes and hydroxylation products obtained therefrom in accordance with the practice of the present invention. 

What is claimed is:
 1. A biologically pure culture having all of the identifying characteristics of the bacterium NRRL-B-18737. 